The goals of the proposed studies are to identify and characterize the rate-limiting step for relaxation in rabbit skeletal muscle fibers and rat cardiac trabeculae by selectively altering each event of relaxation: 1.) lowering of myoplasmic (Ca2+ by the sarcoplasmic reticulum (SR) Ca- ATPase, 2.) dissociation of Ca2+ from the regulatory sites(s) of troponin C (TnC) and 3.) cross-bridge detachment and examining the effect of the alterations on relaxation rate. Aim 1: Directly assess the role of TnC in relaxation induced by flash photolysis of a caged Ca2+ chelator, diazo-2, by determining the effect on relaxation rates of: a) functional TnC mutants which exhibit a 10-fold difference in Ca2+ affinities and off-rates, and b) phosphorylation of cardiac TnI. Aim 2: Determine the effect of increasing (with intracellular Ca2+ chelators) and decreasing (with selective inhibitors of the SR Ca-ATPase) the rate of Ca2+ sequestration on the relaxation rate of intact cardiac trabeculae. Aim 3: Evaluate the contribution of cross-bridge cycling rate and number of cross-bridges to relaxation kinetics by determining the rates of diazo-2 induced relaxation in: a) fast-twitch psoas and slow-twitch soleus fibers, and b) in cardiac muscle from rats which have been treated to produce 100 % V1 or V3 myosin isoforms and by altering the number of force generating cross-bridges by varying Ca2+ concentration or by vanadate. Aim 4: Separate the effect of phosphorylation of phospholamban from phosphorylation of TnI on relaxation rate in cardiac muscle. Twitch-like contractions will be induced in skinned trabeculae with functionally intact SR by flash photolysis of a caged Ca2+. Relaxation rate after phosphorylation of phospholamban and TnI will be compared to relaxation rate when Ca2+ off- rate from TnC is accelerated by a TnC mutant, thus mimicking the effect of TnI phosphorylation. Selectively altering rates of Ca2+ sequestration, Ca2+ dissociation from TnC and cross-bridge kinetics and number will allow determination of the rate-limiting step for relaxation under various conditions.